Antibiotic A43F

ABSTRACT

The antibiotic A43F is active against a variety of plant pathogenic fungi. The antibiotic A43F is produced by growing an as yet unclassified species of fungus (ATCC No. 20529) on suitable fermentation media and isolating the bioactive component therefrom.

SUMMARY OF THE INVENTION

This invention relates to a new antifungal antibiotic agent. More particularly, the instant invention relates to a new antifungal antibiotic agent hereinafter referred to as A43F. The invention encompasses the antibiotic in dilute forms, as crude concentrates and in pure forms.

It is an object of the instant invention to provide a new and useful antifungal antibiotic which is highly effective in inhibiting the growth of a wide variety of plant pathogens. Another object is to provide a process for preparing the novel antibiotic substance by the fermentation of a nutrient media with an as yet unclassified species of fungus initially isolated from a fluff sample taken from a chicken incubator. Other objects will be apparent from the detailed description of the instant invention hereinafter provided.

In its composition of matter aspect, therefore, the instant invention may be described as residing in the concept of a novel antibiotic characterized by having the following structural formula: ##STR1## This structure has been assigned to antibiotic A43F on the basis of various observed physical and chemical characteristics hereinafter described. The structure has not been determined with certitude, however, and it is to be understood that the structure presented above represents a working hypothesis merely.

Antibiotic A43F inhibits the growth of a variety of fungi which are pathogenic to plant life including Puccinia recondita f. sp. tritici, Piricularia oryzae, Phytophthora infestans, Alternaria solani and Erysiphe polygoni. It is contemplated, therefore, that antifungally effective amounts of antibiotic A43F will be applied as an agricultural fungicide in the treatment and control of pathogenic fungus infestations of plants. The antifungal activity of antibiotic A43F has been confirmed in vivo employing standard greenhouse bioassay techniques.

As pointed out above, antibiotic A43F is prepared by growing under controlled conditions an as yet unclassified species of fungus initially isolated from a fluff sample taken from a chicken incubator. This fungus species has been designated MF 4683 in the culture collection of MERCK & CO., Inc., Rahway, New Jersey. A viable culture thereof has been placed on permanent deposit with the culture collection of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, and has been assigned accession number ATCC No.20529.

CHEMICAL AND PHYSICAL CHARACTERISTICS OF ANTIBIOTIC A43F

Antibiotic A43F is a white amorphous solid of elemental composition C₄₅ H₇₁ N₇ O₁₁. Molecular weight 885 was determined by high-resolution mass spectrometric measurement at m/e 867 (M-H₂ O): found 867.5056, calculated 867.5106. The molecular weight 885 was determined by field desorption mass spectrometry.

Ultraviolet absorption of antibiotic A43F was measured in neutral and acid (0.01 N HCl) methanol and in basic (0.01 N NaOH)methanol.

Neutral and Acid Methanol

λ max 225 nm, E^(1%) =230;

λ max 278 nm, E^(1%) =38;

λ max 284 nm, (sh.), E^(1%) =33.

Basic Methanol

λ max 245 nm, E^(1%) =200;

λ max 294 nm, E^(1%) =50.

The infrared spectrum of antibiotic A43F in KBr is shown in FIG. 1.

The ¹ H NMR spectrum of antibiotic A43F is shown in FIG. 2. Chemical shifts are given in ppm (parts-per-million) relative to internal tetramethylsilane. The solvent is CDCl₃.

¹³ C NMR spectra were recorded in CDCl₃ : CD₃ OD (4:1) at a concentration of 60 mg./0.45 ml. Chemical shifts for all 45 carbon atoms are given in the following list in ppm relative to internal tetramethylsilane. Signals representing more than one carbon atom are indicated by the number of carbon atoms in brackets after the chemical shift figure.

14.1, 16.0, 18.3, 19.1, 20.4, 21.2, 22.8, 24.8, 25.6, 27.0, 29.5⁻, 29.5⁺, 29.7 [3], 30.1, 3.20, 32.6, 33.0, 34.6, 35.9, 37.9, 47.8, 48.1, 53.9, 55.8, 58.0, 59.8, 61.6, 67.7, 74.8, 115.7 [2], 127.9, 130.4 [2], 156.0, 171.7, 171.9, 172.4 [2], 172.9, 173.0, 174.6, 176.2 ppm.

Antibiotic A43F contains on equivalent each of L-valine, L-glutamine, L-proline, D-alanine, D-tyrosine, D-allothreonine and δ-hydroxymyristic acid.

The structure of antibiotic A43F (as determined by high-resolution mass spectrometry, ¹ H and ¹³ C NMR and IR spectroscopy, amino acid analysis of the antibiotic and its tyrosine O-methyl ether derivative is shown below and in formula I above.

PRODUCTION OF ANTIBIOTIC A43F--FERMENTATION PROCESS

The standard operating procedure for the production of antibiotic A43F by fermentation of the fungus ATCC No. 20529 is described below.

1. "A" Stage

The culture (ATCC No. 20529) is maintained in frozen vials at -70° to -90° C. To obtain the frozen vegetative stage, the culture is grown in seed media at 28° C., 220 rpm for one day. 20% glycerol is added and the mixture subdivided into 2 ml aliquot per vial. The vials are asceptically sealed and stored in the gaseous phase of liquid nitrogen.

2. "B" Stage

Vessel: 250 ml 3 baffled Erlenmeyer flask with 50 ml. per flask.

Medium:

    ______________________________________                                                               WT/VOL                                                   ______________________________________                                         Corn steep liquor     0.5%                                                     Tomato paste          4.0%                                                     Oat flour No. 36      1.0%                                                     Glucose               1.0%                                                     *Trace element mix No. 2                                                                             1.0% vol/vol                                             ______________________________________                                         *Trace element mix No. 2 (Final "B" Stage                                      Medium Concentration)                                                                                mgm/1                                                    ______________________________________                                         FeSO.sub.4 . 7H.sub.2 O                                                                              10.0                                                     MnSO.sub.4 . 4H.sub.2 O                                                                              10.0                                                     CuCl.sub.2 . 2H.sub.2 O                                                                              0.25                                                     CaCl.sub.2            1.0                                                      H.sub.3 BO.sub.3      0.56                                                     (NH.sub.4).sub.6 Mo.sub.7 O.sub.24 . 4H.sub.2 O                                                      0.19                                                     ZnSO.sub.4 . 7H.sub.2 O                                                                              2.0                                                      Pre-sterile pH        6.8                                                      ______________________________________                                    

Inoculum: 2 ml from frozen vial.

Incubation: 24 hours at 150 rpm and 28° C.

Sterility: Streak plates and gram stain.

3. "C" Stage

Vessel: 2 liter 3 baffled Erlenmeyer flask with 500 ml.

Media: Same as "B" stage.

Inoculum: 10 ml. from "B" stage.

Incubation: 24 hours at 150 rpm and 28° C.

Sterility: Streak plates and gram stain.

4. "D" Stage

Vessel: 50 gallon stainless steel fermentor.

Medium:

    ______________________________________                                                             WT/VOL                                                     ______________________________________                                         Corn steep liquor     0.5%                                                     Tomato paste          4.0%                                                     Oat flour             1.0%                                                     Dextrose              1.0%                                                     FeSO.sub.4 . 7H.sub.2 O                                                                              0.001%                                                   MnSO.sub.4 . 4H.sub.2 O                                                                              0.001%                                                   CuCl.sub.2 . 2H.sub.2 O                                                                              0.000025%                                                CaCl.sub.2 . 2H.sub.2 O                                                                              0.0001%                                                  H.sub.3 BO.sub.3      0.000056%                                                (NH.sub.4).sub.6 Mo.sub.7 O.sub.24 . 4H.sub.2 O                                                      0.000019%                                                ZnSO.sub.4 . 7H.sub.2 O                                                                              0.2%                                                     Polyglycol 2000       50 ml                                                    Pre-sterile pH        6.8                                                      ______________________________________                                    

Sterilization: 15 minutes at 121° C.

Inoculum: 1 liter "C" stage.

Fermentor Volume A.I.: 161 liters.

Cycle Temperature: 28° C.

Airflow: 3 CFM.

Agitation: 150 rpm.

pH control: None.

Cycle time: 24 hours.

Defoamer: None.

Cycle Parameters: pH/12 hours.

    ______________________________________                                                Batch Age (hrs.)                                                                          pH                                                           ______________________________________                                                AI         6.8                                                                  0         6.6                                                                 12         6.2                                                          ______________________________________                                    

5. "E" Stage

Vessel: 200 gallon stainless steel fermentor.

Medium:

    ______________________________________                                                            WT/VOL                                                      ______________________________________                                         Sucrose               6%                                                       Bacto peptone         1%                                                       Ardamine pH           0.2%                                                     Pre-sterile pH        6.0                                                      ______________________________________                                    

Sterilization: 15 minutes at 121° C.

Inoculum: 43 liters "D" stage.

Fermentor Volume A.I.: 510 liters.

Cycle Temperature: 24° C.

Airflow: 10 CFM.

Agitation: 130 rpm.

pH Control: None.

Cycle time: 96 hours.

Defoamer: None.

Cycle parameters: pH/12 hours.

Final Activity: 23 mm diameter zone (1/2 inch disc whole broth) by Cochliobolus miyabeanus (ATCC No. 11608).

    ______________________________________                                         Batch Age (hrs.)     pH                                                        ______________________________________                                          0                   6.3                                                       12                   6.0                                                       24                   4.9                                                       36                   5.0                                                       48                   4.9                                                       60                   4.8                                                       72                   4.7                                                       84                   4.7                                                       96                   4.6                                                       ______________________________________                                    

    ______________________________________                                         Seed Media for "A" Stage                                                       ______________________________________                                         Corn Steep Liquor      5      grams                                            Tomato Paste           40     grams                                            Cerelose               10     grams                                            *Trace Element Mix No. 2                                                                              10     ml                                               Distilled H.sub.2 O pH 6.8                                                                            1000   ml                                               ______________________________________                                         *Trace Element Mix No. 2 contains the following                                ingredients:                                                                   ______________________________________                                         FeSO.sub.4 . 7H.sub.2 O                                                                              1000.0  mg/L                                             MnSO.sub.4 . 4H.sub.2 O                                                                              1000.0  mg/L                                             CuCl.sub.2 . 2H.sub.2 O                                                                              25.0    mg/L                                             CaCl.sub.2 . 2H.sub.2 O                                                                              100.0   mg/L                                             H.sub.3 BO.sub.3      56.0    mg/L                                             (NH.sub.4).sub. 6 Mo.sub.7 O.sub.24 . 4H.sub.2 O                                                     19.0    mg/L                                             ZnSO.sub.4 . 7H.sub.2 O                                                                              200.0   mg/L                                             Distilled H.sub.2 O   1000.   ml                                               ______________________________________                                    

Applicants also have found that antibiotic A43F readily may be produced by static fermentation of the fungus ATCC No. 20529 and that this static fermentation process is preferred where it is desired to achieve increased titer of antibiotic A43F in the fermentation broth. The static fermentation may be carried out by aseptically transferring the contents of a nitrogen frozen vial (2 ml) of the fungus ATCC No. 20529 to a 250 ml. unbaffled flask containing 40 ml. of seed medium (Seed Media for "A" Stage described above). The flask then is incubated with agitation for 24 hours at 28° C. at 220 R.P.M. After incubation 1 ml of inoculum is aseptically transferred to a 250 ml. unbaffled flask containing 40 ml of production medium and allowed to incubate statically for 4 days at 25° C. The constituents for the production medium are:

    ______________________________________                                         Sucrose              60     grams                                              Bacto-peptone        10     grams                                              yeast autolysate                                                                (ardamine pH)       2      grams                                              distilled H.sub.2 O  1000   ml                                                 pH 6.0                                                                         ______________________________________                                    

Upon harvest and assay, the level of antibiotic A43F present has been determined to be approximately 30 μg/ml. By comparison, shake flasks contain less than approximately 1 μg/ml.

ISOLATION OF ANTIBIOTIC A43F

Antibiotic A43F conveniently is isolated from the fermentation broth described above by techniques illustrated in the following flow sheet: ##STR3##

The isolation illustrated above has been carried out as described below.

Step 1

One volume of methanol was added to 50 gallons of the whole broth obtained from the pilot plant fermentation batch described above ("E"Stage) and the mixture was stirred for one hour at room temperature. This step serves two functions: (1) it renders the whole broth filterable and (2) it extracts the antifungal activity from the cells to the filtrate. Super-cel was added to the mixture with stirring and the resultant slurry was filtered through a Super-cel pad.

Step 2

The filtrate was concentrated to one-tenth the volume of the whole broth and extracted two times with an equal volume of ethyl acetate. The combined ethyl acetate layers were concentrated to 1-2 l., dried with sodium sulfate and filtered. Dry weight measurement showed 128 gm. solids at this stage.

Step 3

The ethyl acetate concentrate was chromatographed on 4.5 l. of silica gel formed in ethyl acetate. Elution with ethyl acetate and increasing volumes of methanol in ethyl acetate was made while collecting fractions of 2-3 l. Bioactivity in the effluent was monitored by agar diffusion assays against Cochliobolus miyabeanus (ATCC No. 11608). Bioactivity appeared as a rather sharp fraction eluting with 30% methanol in ethyl acetate and containing 5.1 gm. of solids.

Step 4

The active fraction (4.6 gm.) from above was rechromatographed on 1.4 l. of silica gel formed in methanol and subsequently washed with acetone and ethyl acetate. The sample was loaded in ethyl acetate and the column eluted with ethyl acetate containing increasing amounts of methanol. Fractions of one liter were collected and assayed for bioactivity as above. Two bioactive fractions eluted with 20% methanol in ethyl acetate were obtained containing 676 mg. and 476 mg. solids, respectively.

Step 5

Both active fractions were purified to homogeneity using Sephadex LH-20 column chromatography as described below. Sephadex LH-20 is a hydroxypropylated dextran gum and is supplied in the form of a dry, free-running powder by Pharmacia Fine Chemicals, Inc., Piscataway, New Jersey.

A 290 mg. sample obtained from the silica gel chromatography described above was dissolved in methylene chloride:hexane:methanol (10:10:1,2 ml.) and chromatographed on an Sephadex LH-b 20 column (150 ml.) equilibrated in the same solvent system. The fractions (8 ml.) were assayed for bioactivity as before and also examined by silica gel thin layer chromatography using the upper phase of the solvent system, butanol:acetic acid:water (5:1:3), as developer. Fractions 33-40 contained the bioactivity as well as a single spot (R_(f) 0.44) on TLC examination (iodine vapors, UV light, water spray). These fractions were pooled, evaporated to dryness and freezedried from dioxane to give 108 mg. of pure antibiotic A43F.

Antibiotic A43F is isolated from the static culture described above by techniques similar to those already described above. For example, 8,400 ml of a static culture was decanted to remove the liquid portion and the mycelia (about 1080 gm., wet weight) were ground in a Waring blender with 1200 ml. of methanol for about 2 minutes. After addition of one volume of methanol to the homogenate, it was stirred for 4 hours and allowed to stand at room temperature overnight. After filtration, the methanol was removed by distillation in vacuo. The resulting aqueous mileau was brought to 500 ml. with water and extracted two times with 1000 ml. of ethyl acetate. The combined ethyl acetate extracts then are treated as described in Steps 3-5, above, in order to obtain pure antibiotic A43F.

ANTIFUNGAL ACTIVITY OF ANTIBIOTIC A43F

In order to evaluate the antifungal activity of antibiotic A43F, the antibiotic was tested in vivo against a variety of plant pathogenic fungi employing standard green house bioassay techniques. The antibiotic was tested at three levels, 1000 PPM, 500 PPM, and 100 PPM (PPM=parts per million) against various fungal infections in the greenhouse including wheat leaf rust, rice blast, late blight of tomato, early blight of tomato and powdery mildew of bean as a protectant (treat and infect same leaf surface) and, similarly as a systemic (treat and infect different leaf surfaces or examine the ability to protect newly-emerging leaves). These tests were carried out as follows:

Leaf Rust of Wheat

Pathogen: Puccinia recondita f. sp. tritici

Host: Wheat, variety Yorkstar

Growth Conditions: Wheat plants grown to the one leaf stage in the greenhouse on Swiss Farm potting soil in 7 ounce styrafoam pots. Thinned to 3 seedlings per pot. Day temperatures 20°-28° C.; night temperatures 15°-20° C. Soluble 20--20--20 fertilizer applied weekly.

Treatment: Test solutions or suspensions of candidate compounds sprayed on both leaf surfaces to run-off with a No. 152 DeVilbiss atomizer at 10 psi. Formulations are prepared by dissolving compounds in methanol-Tween 20 mixture and bringing to volume with deionized water.

Inoculation: Two days after treatment a uredospore suspension (20,000/ml.) is sprayed on both leaf surfaces with a No. 152 DeVilbiss atomizer at 10 psi. The inoculum is sprayed to a point just before run-off (non-coalescing droplets). The inoculated plants are maintained at 100 percent relative humidity and 21° C. for 24 hours.

Incubation: The treated and inoculated plants are incubated in the greenhouse (15°-20° C. night temperatures; 20°-28° C. day temperatures) until symptoms appear.

Standard of Comparison: Maneb: [[1,2-Ethanediylbis[carbamodithioato]]-(2-)]manganese.

Disease index: number of lesions per basal leaf/leaf area

Literature Citation: Davis, D., L. Chaiet, J. W. Rothrock, J. Deak, S. Halmos, and J. D. Garber, 1960. Chemotherapy of Cereal Rusts with a New Antibiotic. Phytopathology 50: 841-843.

Rice Blast

Pathogen: Piricularia oryzae

Host: Rice, variety Calusa No. 2.

Growth Conditions: Rice plants grown to 5 leaf stage in greenhouse on Swiss Farm Potting soil in 7 ounce styrafoam pots. Thinned to 3 seedlings per pot. Day temperature 20°-28° C.; night temperature 15°-20° C. Soluble 20--20--20 fertilizer applied weekly.

Treatment: Test solutions or suspensions of candidate compounds sprayed on both leaf surfaces to run-off with a No. 152 DiVilbiss atomizer at 10 psi. Formulations are prepared by dissolving compounds in methanol-Tween 20 mixture and bringing to volume with deionized water.

Inoculation: Two days after treatment a spore suspension (50,000/ml) is sprayed on the upper leaf surface with a No. 152 DeVilbiss atomizer at 10 psi. The inoculum is sprayed to a point just before run-off (non-coalescing droplets). The inoculated plants are maintained at 100 percent relative humidity and 28° C. for 24 hours.

Incubation: The treated and inoculated plants are incubated in the greenhouse (15°-20° C. night temperatures; 20°-28° C. day temperatures) until symptoms appear.

Standard of Comparison: Hinosan: O-Ethyl 5,5-diphenylphosphorodithioate.

Disease index: number of lesions per 4th leaf/leaf area

Literature Citation: Kahan, R. P. and J. L. Libby. 1958. The Effect of Environmental Factors and Plant Age on Infection of Rice Blast Fungus, Piricularia oryzae Phytopathology 48: 25-30.

Late Blight of Tomato

Pathogen: Phytophthora infestans

Host: Tomato, variety Bonny Best

Growth Conditions: Tomato plants grown to the 5 leaf stage in greenhouse on Swiss Farm potting soil in 7 ounce styrafoam pots. Day temperature 20°-28° C.; night temperature 15°-20° C. Soluble 20--20--20 fertilizer applied weekly.

Treatment: Test solutions or suspensions of candidate compounds sprayed on both leaf surfaces to run-off with a No. 152 DeVilbiss atomizer at 10 psi. Formulations are prepared by dissolving compounds in methanol-Tween 20 mixture and bringing to volume with deionized water.

Inoculation: Two days after treatment a swarmspore suspension (10,000/ml) is sprayed on the lower leaf surface with a No. 152 DeVilbiss atomizer at 10 psi. The inoculum is sprayed to a point just before run-off (non-coalescing droplets). The inoculated plants are maintained at 100 percent relative humidity and 17° C. for 24 hours.

Incubation: The treated and inoculated plants are incubated in the greenhouse (15°-20° C. night temperatures; 20°-28° C. day temperatures) until symptoms appear.

Standard of Comparison: Maneb

Disease Index: percent of 3rd, 4th and 5th leaf necrotic

Literature Citation: McCallen, S. E. A. and R. H. Wellman 1943. A Greenhouse Method of Evaluating Fungicides by Means of Tomato Foliage Diseases. Contrib. Boyce Thompson Instit. 13: 93-134.

Early Blight of Tomato

Pathogen: Alternaria solani

Host: Tomato, variety Bonny Best

Growth Conditions: Tomato plants grown to the five leaf stage in greenhouse on Swiss Farm potting soil in 7 ounce styrafoam pots. Day temperature 20°-28° C.; night temperatures 15°-20° C. Soluble 20--20--20 fertilizer applied weekly.

Treatment: Test solutions or suspensions of candidate compounds sprayed on both leaf surfaces to run-off with a No. 152 DeVilbiss atomizer at 10 psi. Formulations are prepared by dissolving compounds in methanol-Tween 20 mixture and bringing to volume with deionized water.

Inoculation: Two days after treatment a spore suspension (10,000/ml) is sprayed on the lower leaf surface with a No. 152 DeVilbiss atomizer at 10 psi. The inoculum is sprayed to a point just before run-off (non-coalescing droplets). The inoculated plants are maintained at 100 percent relative humidity and 21° C. for 24 hours.

Incubation: The treated and inoculated plants are incubated in the greenhouse (15°-20° C. night temperatures; 20°-28° C. day temperatures) until symptoms appear.

Standard of Comparison: Maneb

Disease Index: (number lesions/terminal leaflet of 3rd, 4th, 5th leaf)/leaf area

Literature Citation: McCallen, S. E. A. and R. H. Wellman, 1943. A Greenhouse Method of Evaluating Fungicides by Means of Tomato Foliage Diseases. Contrib. Boyce Thompson Instit. 13: 93-134

Powdery Mildew of Bean

Pathogen: Erysiphe polygoni

Host: Bean, variety Bountiful

Growth Conditions: Bean plants grown in greenhouse to point where the primary leaves are two-thirds expanded. The plants are grown on Swiss Farm potting soil in 7 ounce styrafoam pots. Dry temperatures 20°-28° C.; 15°-20° C. night temperatures. Soluble 20--20--20 fertilizer applied at weekly intervals.

Treatment: Test solutions or suspensions of candidate compounds sprayed on both leaf surfaces to run-off with a No. 152 DeVilbiss atomizer at 10 psi. Formulations are prepared by dissolving compounds in methanol-Tween 20 mixture and bringing to volume with deionized water.

Inoculation: Two days after treatment a spore suspension (5,000/ml) is sprayed on the upper leaf surface with a No. 152 DeVilbiss atomizer at 10 psi. The inoculum is sprayed to a point just before run-off (non-coalescing droplets).

Incubation: The treated and inoculated plants were incubated in the growth room (12° C. night; 18° C. day temperatures).

Standard of Comparison: Benomyl-[1-[(Butylamino)carbonyl]-1H-benzimidazol-2-yl]carbamic acid methyl ester

Disease Index: number of lesions per primary leaf/leaf area

Literature Citation: El-Zayat, R. J. Lukens, A. E. Dimond and J. G. Horsfall. 198. Systemic Action of Nitrophenols Against Powdery Mildew. Phytopathology 58: 434-437. Methods and Materials

1. Protectant bioassays: As described above.

2. Systemic bioassay: Same procedure as in protectant bioassay except as indicated below.

a. Wheat leaf rust--leaf emerging was inoculated 5 days after treatment.

b. Rice blast--leaf emerging was inoculated 9 days after treatment.

c. Late blight--lower leaf surface inoculated 2 days after treating upper leaf surface.

d. Early blight--lower leaf surface inoculated 2 days after treating upper leaf surface.

e. Powdery mildew--lower leaf surface inoculated after treating upper leaf surface.

Results

The results of all tests are given in Table 1 below. The untreated controls, test samples and positive controls are presented.

                                      TABLE 1                                      __________________________________________________________________________                                   Late Blight (Tomato)                                                                      Early Blight (Tomato)                                                                     Bean                               Wheat Leaf Rust                                                                           Rice Blast Phytophthora                                                                              Alternaria Powdery Mildew             Treat-  Pustules/Leaf                                                                             Lesions/5th Leaf                                                                          % Leaf Necrosis                                                                           Lesions/Leaf                                                                              Mildew                                                                         Centers/16cm.sup.2         ment PPM                                                                               Protectant                                                                           Systemic                                                                            Protectant                                                                           Systemic                                                                            Protectant                                                                           Systemic                                                                            Protectant                                                                           Systemic                                                                            Protectant                                                                           Systemic             __________________________________________________________________________     Un-  -- 27    42   52    69   84    88   63    70   24    25                   treated                                                                        A43F 1000                                                                              0      5*  <1    1    8     32   14    58   <1    12                        500                                                                               <1    15*  0     9    8     60   29    45   <1    14                        100                                                                               6     31*  <1    26   33    88   38    46   7     17                                 Car-                  --         --                                      Maneb boxin**                                                                             Hinosan                                                                              Hinosan                                                                             Maneb --   Maneb --   Benomyl                                                                              Benomyl                   1000                                                                              --    4    --    31   --    --    4    --   --     0                        100                                                                               0     --   < 1   --   11    --   25    --   0      7                        10 12    --   8     --   63    --   58    --   10    --                        1  20    --   16    --   77    --   --    --   26    --                   __________________________________________________________________________       *Phytotoxicity                                                                 **5,6Dihydro-2-methyl-N-phenyl-1,4-oxathiin-3-carboxamide               

From the foregoing table it appears that antibiotic A43F is:

(a) highly active against wheat leaf rust, rice blast and powdery mildew as protectants; equal to or approaching its standards of comparison as a protectant;

(b) moderately active against tomato early and late blight as a protectant;

(c) shows good activity against wheat leaf rust, rice blast and powdery mildew as a systemic and moderate activity against tomato late blight;

and that it shows very low or not evident phytotoxicity on wheat, rice, tomato and bean.

Similar tests for protectant activity of antibiotic A43F at lower concentrations, against powdery mildew of bean and rice blast are shown in Table 2 below:

    ______________________________________                                         Rice Blast                                                                     Compound  Concentration                                                                              Lesions                                                  No.       (PPM)*      per 5th Leaf                                                                              Remarks                                       ______________________________________                                         A43F      10          2          no injury                                               1           4          no injury                                               0.1         8          no injury                                               0.01        7          no injury                                     Untreated                                                                      control   --          22         no injury                                     Benomyl   100         0.1        no injury                                               10          4          no injury                                               1           4          no injury                                     ______________________________________                                           *compounds dissolved in methanol and diluted with water containing Tween      20.                                                                      

It should be understood that antibiotic A43F may be utilized in diverse anti-fungal formulations; solid, including finely divided powders and granular materials, as well as liquid, such as solutions, emulsions, suspensions, concentrates, emulsifiable concentrates, slurries and the like depending upon the application intended and the formulation media desired. Thus, it will be appreciated that antibiotic A43F may be employed to form anti-fungal compositions containing antibiotic A43F as the essential active ingredient thereof which compositions may also include finely divided dry or liquid diluents, extenders, fillers, conditioners and excipients, including various clays, diatomaceous earth, talc or the like or water and various organic liquids such as lower alkanols, for example, methanol, ethanol and isopropanol, and or kerosene, benzene, toluene and other petroleum distillate fractions or mixtures thereof. 

The subject matter which applicants regard as their invention is particularly pointed out and distinctly claimed as follows:
 1. The antibiotic A43F having the following structural formula: ##STR4##
 2. A method of controlling fungus growth which comprises contacting the area upon which fungus growth is to be controlled with an effective amount of antibiotic A43F.
 3. A method of controlling plant pathogenic fungus growth which comprises contacting plant surfaces upon which fungus growth is to be controlled with an effective amount of antibiotic A43F.
 4. A method of controlling plant pathogenic fungus growth which comprises treating a plant in which fungus growth is to be controlled with an effective amount of antibiotic A43F.
 5. A anti-fungal composition which comprises an effective quantity of antibiotic A43F and a carrier therefor. 